Methods of protecting against apoptosis using lipopeptides

ABSTRACT

The use of lipopeptides comprising variable groups defined as H or optionally substituted C 6 -C 20  aliphatic and a peptide of one of the following amino acid sequences: GEESN (SEQ ID NO: 17), QGEESNDK (SEQ ID NO: 20), and VQGEESNDK (SEQ ID NO: 21). The use of the lipopeptides for activating Toll-like receptors (TLRs) in cells is described.

FIELD OF THE INVENTION

This invention relates to the use of inducers of NF-κB to protectmammals from the effects of apoptosis. More specifically, this inventionrelates to the use of inducers of NF-κB to protect mammals from exposureto stress, such as radiation and cancer treatments.

BACKGROUND OF THE INVENTION

The progression from normal cells to tumor cells involves a loss ofnegative mechanisms of growth regulation, including resistance to growthinhibitory stimuli and a lack of dependence on growth factors andhormones. Traditional cancer treatments that are based on radiation orcytotoxic drugs rely on the differences in growth control of normal andmalignant cells. Traditional cancer treatments subject cells to severegenotoxic stress. Under these conditions, the majority of normal cellsbecome arrested and therefore saved, while tumor cells continues todivide and die.

However, the nature of conventional cancer treatment strategies is suchthat normal rapidly dividing or apoptosis-prone tissues are at risk.Damage to these normal rapidly dividing cells causes the well-known sideeffects of cancer treatment (sensitive tissues: hematopoiesis, smallintestine, hair follicles). The natural sensitivity of such tissues iscomplicated by the fact that cancer cells frequently acquire defects insuicidal (apoptotic) machinery and therapeutic procedures that causedeath in normal sensitive tissues may not be effective on cancer cells.Conventional attempts to minimize the side effects of cancer therapiesare based on (a) making tumor cells more susceptible to treatment, (b)making cancer therapies more specific for tumor cells, or (c) promotingregeneration of normal tissue after treatment (e.g., erythropoietin,GM-CSF, and KGF). Each of these, however, has limited effectiveness. Asa result, there continues to be a need for therapeutic agents tomitigate the side effects associated with chemotherapy and radiationtherapy in the treatment of cancer. This invention fulfills these needsand provides other related advantages.

SUMMARY OF THE INVENTION

Provided herein is a method of protecting a mammal from one or moreconditions or treatments that trigger apoptosis. A mammal may beadministered a composition comprising a pharmaceutically acceptableamount of a compound of the formula:

wherein,

-   -   R₁ represents H or —CO—R₄,    -   R₂, R₃ and R₄ independently are H or optionally substituted        C₈-C₁₆ aliphatic;    -   X is a peptide; and    -   Z is S or CH₂.

The peptide may comprise a sequence set forth in SEQ ID NOs: 1-52. Thefirst five amino acids of the peptide may be chosen from the amino acidsat the positions referred to in Table 2. The compound may be an RR or RSstereoisomer, or mixture thereof. The compound may also be of theformula:

The condition that triggers apoptosis may be radiation, wounding,poisoning, infection or temperature shock. The treatment that triggersapoptosis may be a cancer treatment. The cancer treatment may bechemotherapy or radiation therapy. The tissue wherein apoptosis istriggered may be the spleen, thymus, GI tract, lungs, kidneys, liver,cardiovascular system, blood vessel endothelium, nervous system (centralor peripheral), hematopoietic progenitor cells (bone marrow), immunesystem, hair follicles, or the reproductive system.

The compound may be administered in combination with a radioprotectant.The radioprotectant may be an antioxidant, such as amifostine or vitaminE. The radioprotectant may also be a cytokine, such as stem cell factor.The radioprotectant may also be flagellin, latent TGFβ, or an activatorof a TLR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-D illustrate that p53 deficiency accelerated the development ofradiation-induced gastrointestinal syndrome in mice. FIG. 1A presentsgraphs of the percent survival of mice exposed to 9, 12.5, 25, or 5×2.5Gy of total body gamma radiation following pretreatment with aninhibitor of p53, pifithrin-alpha (PFT), or DMSO (control). p53-nullmice were also exposed to the fractioned cumulative radiation dose of12.5 Gy (5×2.5 Gy). FIG. 1B presents graphs of the percent survival ofwild type and p53-null mice after exposure to low (10 Gy) or high (15Gy) doses of total body gamma radiation. FIG. 1C presents a graphillustrating the percent survival of mice exposed to 15 Gy of total bodygamma radiation following reconstitution with bone marrow (BM) from wildtype or p53-null mice. FIG. 1D presents haematoxylin-eosin stainedparaffin intestinal sections from wild type and p53-null mice at theindicated time points after 15 Gy of gamma radiation. Insets at 24 hshow TUNEL staining of crypt regions.

FIGS. 2A-C illustrate the dynamics of cell proliferation and survival inthe small intestines of wild type and p53-null mice. FIG. 2A (left)shows autoradiographs of whole-body sections of wild type and p53-nullmice injected with ¹⁴C-thymidine that were treated with 15 Gy of gammaradiation or not treated. Arrows point to the intestines. FIG. 2A(right) shows photomicrographs of BrdU incorporation in the smallintestine of wild type and p53-null mice at different time points after15 Gy of gamma radiation. Regions of the 96 h images are shown at highermagnification. FIG. 2B presents a graph of the number of BrdU positivecells/crypt in the small intestine of wild type and p53-null mice atdifferent time points after 15 Gy of gamma radiation. FIG. 2C presentsphotomicrographs of BrdU-labeled cells in the small intestine of wildtype and p53-null mice at different time points after 15 Gy of gammaradiation. BrdU was injected 30 min before irradiation and the mice weresacrificed at the indicated time points.

FIG. 3 illustrates the radioprotective effect of the compound, CBLB601.Shown are graphs of the percent of survival of mice exposed to 9, 12.5,25, or 5×2.5 Gy of total body gamma radiation following pretreatmentwith CBLB601 or PBS.

FIG. 4 illustrates alterations in spleen size after exposure to 13 Gy oftotal body gamma irradiation following pre treatment with CBLB601 orPBS. On the left is a graph of spleen weights of PBS and CBLB601-treatedmice, and on the right are images of spleens from the control or treatedmice.

FIGS. 5A-B illustrate the determination of the optimal time forintraperitoneal injection of CBLB601. FIG. 5A shows a graph of thepercent survival of mice exposed to 10 Gy of total body irradiation(TBI) following intraperitoneal administration of PBS or CBLB601 24, 6,1 or 0.5 hr prior to irradiation. FIG. 5B shows a graph of the percentsurvival of mice exposed to 10 Gy of TBI following intraperitonealadministration of PBS or CBLB601 96, 72, 48, 24 or 1 hr prior toirradiation.

FIGS. 6A-B illustrate the determination of the optimal dose of CBLB601.FIG. 6A shows a graph of the percent survival of mice exposed to 10 Gyof TBI following intraperitoneal administration of PBS or 1, 3, 10, 20,30 μg of CBLB601/mouse 24 hr prior to irradiation. FIG. 6B shows a graphof the percent survival of mice exposed to 10 Gy of TBI followingintraperitoneal administration of PBS or 0.1, 0.3, 1, 3, 10, or 15 μg ofCBLB601/mouse 24 hr prior to irradiation.

FIGS. 7A-B illustrate the determination of the dose of radiationprotected by CBLB601. FIG. 7A shows a graph of the percent survival ofmice exposed to 10, 11, 12, 13, 14, or 15 Gy of TBI followingintraperitoneal administration of PBS 24 hr prior to irradiation. FIG.7B shows a graph of the percent survival of mice exposed to 10, 11, 12,13, 14, or 15 Gy of TBI following intraperitoneal administration of 3 μgof CBLB601/mouse 24 hr prior to irradiation.

FIG. 8 illustrates the radioprotective effect of intramuscularadministration of CBLB601. Shown is a graph of the percent survival ofmice exposed to 10 Gy of TBI following intraperitoneal administration ofPBS or intramuscular administration of 1, 3, or 10 μg of CBLB601/mouse24 hr prior to irradiation.

FIG. 9 depicts survival after different doses of radiation and differentdoses of CBLB601. Shown is a graph of the percent survival of miceexposed to 10, 11, or 12 Gy of TBI following or intramuscularadministration of PBS or 0.3, 1, 3, 10 or 30 μg of CBLB601/mouse 24 hrprior to irradiation.

FIG. 10 compares survival after different doses of CBLB601 wereadministered via different routes. Shown is a bar graph of the percentsurvival of mice exposed to 10 Gy of TBI following intraperitoneal orintramuscular administration of different doses of CBLB601 24 hr priorto irradiation.

FIG. 11 compares survival after different doses of CBLB601, expressed asμg/kg, were administered via different routes. Shown is a graph of thepercent survival of mice exposed to 10 Gy of TBI followingintraperitoneal or intramuscular administration of different doses ofCBLB601 24 hr prior to irradiation.

FIGS. 12A-C illustrate the determination of the optimal time forintramuscular injection of CBLB601. FIG. 12A shows a graph of thepercent survival of mice exposed to 10 Gy of TBI following intramuscularadministration of PBS or 3 μg of CBLB601/mouse 24, 6, 3, or 1 hr priorto irradiation or 1 or 3 hr after irradiation. FIG. 12B shows a graph ofthe percent survival of mice exposed to 10 Gy of TBI followingintramuscular administration of PBS or 3 μg of CBLB601/mouse 48, 36, 24,12, or 6 hr prior to irradiation. FIG. 12C shows a graph of the percentsurvival of mice exposed to 10 Gy of TBI following intramuscularadministration of PBS or 1, 3, 10, or 30 μg of CBLB601/mouse 1 hr afterirradiation.

FIG. 13 compares survival as a function of the time of administrationand route of administration of CBLB601. Shown is a bar graph of thepercent survival of mice exposed to 10 Gy of TBI followingintraperitoneal or intramuscular administration of 3 μg of CBLB601/mouseat various times prior to irradiation.

FIG. 14 illustrates the determination of the Dose Modification Factor atday 30 (DMF₃₀) for CBLB601 under the optimal radioprotective conditions.Shown is a graph of the percent survival of mice exposed to variousdoses of radiation following intramuscular administration of PBS or 3 μgof CBLB601/mouse 24 hr prior to irradiation.

FIG. 15 presents a graph of the average weights of spleens fromirradiated control and CBLB601-treated mice. Plotted is the spleenweight per body weight ratio for mice exposed to 0, 6, or 10 Gy of TBIfollowing intramuscular administration of PBS or 3 μg of CBLB601/mouse24 hr prior to irradiation.

FIGS. 16A-C depict the immune responses of CBLB601-treated mice thatwere immunized with flagellin 8, 18, or 20 weeks after irradiation. FIG.16A shows the immune response of the different groups one month afterthe first immunization. FIG. 16B shows the immune response of thedifferent groups one month after the first bleed. FIG. 16C shows thesecondary immune response to flagellin of the different groups 10 daysafter the third immunization.

FIG. 17 is a graph of the activation of a NF-κB reporter by variousdoses of CBLB613 and CBLB601 in 293 cells expressing the TLR2/TLR6heterodimer.

FIGS. 18A-B present the activation of a NF-κB reporter by various CBLBcompounds in 293 cells expressing the TLR2/TLR6 heterodimer. FIG. 18Apresents NF-κB activation by various doses of CBLB601, CBLB612, CBLB614,or CBLB615. FIG. 18B presents NF-κB activation by various doses ofCBLB601, CBLB612, CBLB614, or CBLB615, and no activation by thecorresponding free peptides.

FIG. 19 is a graph of the activation of a NF-κB reporter by variousdoses of CBLB617 and CBLB601 in 293 cells expressing the TLR2/TLR6heterodimer.

FIG. 20 illustrates the radioprotective activity of CBLB613. Shown is agraph of the percent survival of mice exposed to 10 Gy of TBI followingintramuscular administration of PBS or 0.3, 1, 3, 10, 30, or 82.5 μg ofCBLB613/mouse 24 hr prior to irradiation.

FIG. 21 illustrates the radioprotective activity of CBLB612, CBLB614,and CBLB615. Shown is a graph of the percent survival of mice exposed to10 Gy of TBI following intramuscular administration of PBS or variousdoses of CBLB612, CBLB614, or CBLB615 24 hr prior to irradiation.

FIG. 22 illustrated the mitigative activity of CBLB612. Shown is a graphof the percent survival of mice treated with 50 μg of CBLB612/mouse orPBS 1 hr after exposure to 8.5, 9, or 10 Gy of TBI.

DETAILED DESCRIPTION

Provided herein is a method of protecting normal cells and tissues fromapoptosis caused by a variety of stresses. Apoptosis normally functionsto “clean” tissues from wounded or genetically damaged cells, whilecytokines mobilize the defense system of the organism against thestress. However, under conditions of severe injury, both stress responsemechanisms can by themselves act as causes of death. For example,lethality from radiation may result from massive apoptosis occurring inhematopoietic, immune and digestive systems.

There are two major mechanisms controlling apoptosis in the cell: thep53 (pro-apoptotic) and the NF-κB pathway (anti-apoptotic). Bothpathways are frequently deregulated in tumors: p53 may be lost, whileNF-κB may become constitutively active. Hence, inhibition of p53 and/oractivation of NF-κB in normal cells may protect them from death causedby stresses. Such an approach in cancer treatments would not make tumorcells more resistant to treatment because they may already have thesecontrol mechanisms deregulated. This contradicts the conventional viewon p53 and NF-κB, which are considered as targets for activation andrepression, respectively.

As described herein, NF-κB activity may be induced to protect normalcells from apoptosis. By inducing NF-κB activity in a mammal, normalcells may be protected from apoptosis attributable to cellular stress.Once the normal cells recover from the stress, NF-κB activity may berestored to normal levels. By temporarily inducing NF-κB activity, cellsmay be protected from a variety of stresses. This may provide control ofboth inflammatory responses and the life-death decisions of cells frominjured tissues and organs.

The protective role of NF-κB may be mediated by transcriptionalactivation of multiple genes coding for: a) anti-apoptotic proteins thatblock both major apoptotic pathways, b) cytokines and growth factorsthat induce proliferation and survival of hematopoietic and other stemcells, and c) potent ROS-scavenging antioxidant proteins, such as MnSOD(SOD-2). Thus, for example, by transient activation of NF-κB forradioprotection, it may be possible to achieve not only suppression ofapoptosis in cancer patients, but also the ability to reduce the rate ofsecondary cancer incidence because of its simultaneous immunostimulatoryeffects, which, may be achieved if activation of NF-κB is mediated byToll-like receptors.

Another attractive property of the NF-κB pathway as a target is itsactivation by numerous natural factors. Among these, are multiplepathogen-associated molecular patterns (PAMPs). PAMPs are present onlyin microorganisms and are not found in the host organism, arecharacteristic for large groups of pathogens, and cannot be easilymutated. They are recognized by Toll-like receptors (TLRs), the keysensor elements of innate immunity. TLRs act as a first warningmechanism of the immune system by inducing migration and activation ofimmune cells directly or through cytokine release. TLRs are type Imembrane proteins, known to work as homo- and heterodimers. Upon ligandbinding, TLRs recruit MyD88 protein, an indispensable signaling adaptorfor most TLRs. The signaling cascade that follows leads to effectsincluding (i) activation of NF-κB pathway, and (ii) activation of MAPKs,including Jun N-terminal kinase (JNK). Unlike cytokines, many PAMPs havelittle effect besides activating TLRs, and thus, are unlikely to produceside effects. Moreover, numerous TLTs (TLR1-TLR10) are present inhumans. Consistent with their function of immunocyte activation, allTLRs are expressed in spleen and peripheral blood leukocytes, with moreTLR-specific patterns of expression in other lymphoid organs and subsetsof leukocytes. All of the TLRs are also expressed in the endothelial andmucosal epithelial cells of the skin and the respiratory, intestinal,and genitourinary tracts.

1. Definitions

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

The term “administer”, when used to describe the dosage of an agent thatinduces NF-κB activity, means a single dose or multiple doses of theagent.

The term “aliphatic” as used herein refers to an unbranched, branched orcyclic hydrocarbon group, which may be substituted or unsubstituted, andwhich may be saturated or unsaturated, but which is not aromatic. Theterm aliphatic further includes aliphatic groups, which comprise oxygen,nitrogen, sulfur or phosphorous atoms replacing one or more carbons ofthe hydrocarbon backbone.

The term “alkyl” as used herein alone or in combination refers to abranched or unbranched, saturated aliphatic group. Representativeexamples of alkyl groups include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, octyl,decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl and the like.

The term “alkenyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon double bond which may occur at any stable point alongthe chain. Representative examples of alkenyl groups include, but arenot limited to, ethenyl, E- and Z-pentenyl, decenyl and the like.

The term “alkynyl” as used herein alone or in combination refers to abranched or unbranched, unsaturated aliphatic group containing at leastone carbon-carbon triple bond which may occur at any stable point alongthe chain. Representative examples of alkynyl groups include, but arenot limited to, ethynyl, propynyl, propargyl, butynyl, hexynyl, decynyland the like.

The term “analog” when used in the context of a peptide or polypeptide,means a peptide or polypeptide comprising one or more non-standard aminoacids or other structural variations from the conventional set of aminoacids.

The term “antibody” as used herein means an antibody of classes IgG,IgM, IgA, IgD or IgE, or fragments or derivatives thereof, includingFab, F(ab′)₂, Fd, and single chain antibodies, diabodies, bispecificantibodies, bifunctional antibodies and derivatives thereof. Theantibody may be a monoclonal antibody, polyclonal antibody, affinitypurified antibody, or mixtures thereof which exhibits sufficient bindingspecificity to a desired epitope or a sequence derived therefrom. Theantibody may also be a chimeric antibody. The antibody may bederivatized by the attachment of one or more chemical, peptide, orpolypeptide moieties known in the art. The antibody may be conjugatedwith a chemical moiety.

The term “apoptosis” as used herein refers to a form of cell death thatincludes progressive contraction of cell volume with the preservation ofthe integrity of cytoplasmic organelles; condensation of chromatin(i.e., nuclear condensation), as viewed by light or electron microscopy;and/or DNA cleavage into nucleosome-sized fragments, as determined bycentrifuged sedimentation assays. Cell death occurs when the membraneintegrity of the cell is lost (e.g., membrane blebbing) with engulfmentof intact cell fragments (“apoptotic bodies”) by phagocytic cells.

The term “cancer” as used herein means any condition characterized byresistance to apoptotic stimuli.

The term “cancer treatment” as used herein means any treatment forcancer known in the art including, but not limited to, chemotherapy andradiation therapy.

The term “combination with” when used to describe administration of anagent that induces NF-κB activity and an additional treatment means thatthe agent may be administered prior to, together with, or after theadditional treatment, or a combination thereof.

The term “derivative” when used in the context of a peptide orpolypeptide, means a peptide or polypeptide different other than inprimary structure (amino acids and amino acid analogs). By way ofillustration, derivatives may differ by being glycosylated, one form ofpost-translational modification. For example, peptides or polypeptidesmay exhibit glycosylation patterns due to expression in heterologoussystems. If at least one biological activity is retained, then thesepeptides or polypeptides are derivatives according to the invention.Other derivatives include, but are not limited to, fusion peptides orfusion polypeptides having a covalently modified N- or C-terminus,PEGylated peptides or polypeptides, peptides or polypeptides associatedwith lipid moieties, alkylated peptides or polypeptides, peptides orpolypeptides linked via an amino acid side-chain functional group toother peptides, polypeptides or chemicals, and additional modificationsas would be understood in the art.

The term “fragment” when used in the context of a peptide orpolypeptide, may mean a peptide of from about 6 to about 10 amino acidsin length. The fragment may be 6, 7, 8, 9 or 10 amino acids in length.

The term “homolog” when used in the context of a peptide or polypeptide,means a peptide or polypeptide sharing a common evolutionary ancestor.

The term “saturated” as used herein refers to a group where allavailable valence bonds of the backbone atoms are attached to otheratoms.

The term “substituted” as used herein refers to a group having one ormore hydrogens or other atoms removed from a carbon and replaced with afurther group. Substituted groups herein may be substituted with one tofive, or one to three substituents. Representative examples of suchsubstituents include, but are not limited to aliphatic groups, aromaticgroups, alkyl, alkenyl, alkynyl, aryl, alkoxy, halo, aryloxy, carbonyl,acryl, cyano, amino, nitro, phosphate-containing groups,sulfur-containing groups, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, acylamino, amidino, imino, alkylthio, arylthio,thiocarboxylate, alkylsulfinyl, trifluoromethyl, azido, heterocyclyl,alkylaryl, heteroaryl, semicarbazido, thiosemicarbazido, maleimido,oximino, imidate, cycloalkyl, cycloalkylcarbonyl, dialkylamino,arylcycloalkyl, arylcarbonyl, arylalkylcarbonyl, arylcycloalkylcarbonyl,arylphosphinyl, arylalkylphosphinyl, arylcycloalkylphosphinyl,arylphosphonyl, arylalkylphosphonyl, arylcycloalkylphosphonyl,arylsulfonyl, arylalkylsulfonyl, arylcycloalkylsulfonyl, combinationsthereof, and substitutions thereto.

The term “treat” or “treating” when referring to protection of a mammalfrom a condition, means preventing, suppressing, repressing, oreliminating the condition. Preventing the condition involvesadministering a composition of this invention to a mammal prior to onsetof the condition. Suppressing the condition involves administering acomposition of this invention to a mammal after induction of thecondition but before its clinical appearance. Repressing the conditioninvolves administering a composition of this invention to a mammal afterclinical appearance of the condition such that the condition is reducedor maintained. Elimination the condition involves administering acomposition of this invention to a mammal after clinical appearance ofthe condition such that the mammal no longer suffers the condition.

The term “tumor cell” as used herein means any cell characterized byresistance to apoptotic stimuli.

The term “unsaturated” as used herein refers to a group where at leastone available valence bond of two adjacent backbone atoms is notattached to other atoms.

The term “unsubstituted” as used herein refers to a group that does nothave any further groups attached thereto or substituted therefor.

The term “variant” when used in the context of a peptide or polypeptide,means a peptide or polypeptide that differs in amino acid sequence bythe insertion, deletion, or conservative substitution of amino acids,but retains at least one biological activity. For purposes of thisinvention, “biological activity” includes, but is not limited to, theability to be bound by a specific antibody. A conservative substitutionof an amino acid, i.e., replacing an amino acid with a different aminoacid of similar properties (e.g., hydrophilicity, degree anddistribution of charged regions) is recognized in the art as typicallyinvolving a minor change. These minor changes can be identified, inpart, by considering the hydropathic index of amino acids, as understoodin the art (Kyte et al., J. Mol. Biol. 157:105-132, 1982). Thehydropathic index of an amino acid is based on a consideration of itshydrophobicity and charge. It is known in the art that amino acids ofsimilar hydropathic indexes can be substituted and still retain proteinfunction. In one aspect, amino acids having hydropathic indexes of ±2are substituted. The hydrophilicity of amino acids can also be used toreveal substitutions that would result in proteins retaining biologicalfunction. A consideration of the hydrophilicity of amino acids in thecontext of a peptide permits calculation of the greatest local averagehydrophilicity of that peptide, a useful measure that has been reportedto correlate well with antigenicity and immunogenicity. U.S. Pat. No.4,554,101, incorporated herein by reference. Substitution of amino acidshaving similar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. In one aspect, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

2. Lipopeptides

A lipopeptide may be used as an agent to induce NF-κB activity.Lipopeptides are part of the outer membranes of Gram-negative bacteria,Gram-positive bacteria, and mycoplasma. Bacterial lipopeptides have noshared sequence homology, but are characterized by the unusualN-terminal amino acid S-(2,3-dihydroxypropyl)-L-cysteine that isacylated by two or three fatty acids. Bacterial lipopeptides are strongimmune modulators that activate early host responses after infection bysignaling through TLR2-TLR1 or TLR2-TLR6 heterodimers, leading to theactivation of NF-κB and cytokine production. Synthetic analogues of theN-terminal lipopeptides of natural lipopeptides are potent activators ofTLRs and NF-κB, as well as being immunoadjuvants in vivo and in vitro.

The lipopeptide may be a compound of the formula:

wherein,

-   -   R₁ represents H or —CO—R₄,    -   R₂, R₃ and R₄ independently are H or optionally substituted        aliphatic;    -   X is H or a peptide; and    -   Z is S or CH₂.

The lipopeptide may comprise two or three fatty acids. The aliphaticsubstituents of R₂, R₃ and R₄ may comprise from 6 to 20 carbon atoms.R₂, R₃ and R₄ may be C₆-C₂₀ alkyl, C₆-C₂₀ alkenyl, or C₆-C₂₀ alkynyl.Representative examples of alkyl substituents at R₂, R₃ and R₄ includeC₆, C₈, C₉, C₁₀, C₁₂, C₁₄, and C₁₆. Representative examples of alkenylsubstituents at R₂, R₃ and R₄ include C_(10:1) ^(D1 trans), C_(18:1)^(D9), and C_(18:2) ^(D9, 12).

The peptide may comprise between at least 4 or 5 amino acids and no morethan 20, 30 or 40 amino acids. The peptide moiety may be essential foractivity and the activity of the lipopeptide may by modulated by theamino acid sequence, but biological activity may be insensitive to mostpeptide sequences (Spohn et al., Vaccine, 22(19):2494-9, 2004). Thepeptide may comprise a sequence set forth in Table 1, any sequence atleast 80%, 85%, 90%, or 95% identical thereto, or any analog,derivative, fragment, homolog, variant or substitution thereof. Thepeptide may carry a net negative charge.

TABLE 1 Sequence Length SEQ ID NO SNNA  4  1 GSSHH  5  2 KQNVS  5  3 NNSGK  5  4 QPDRY  5  5 RPDRY  5  6 SEEEE  5  7 SKKKK  5  8 SNNNA  5  9SPPPP  5 10 GQHHM  5 11 GQHHH  5 12 SSHHM  5 13 GSHHM  5 14 SQMHH  5 15GETDK  5 16 GEESN  5 17 GEEDD  5 18 TENVKE  6 19 QGEESNDK  8 20VQGEESNDK  9 21 FEPPPATTT  9 22 GDKYFKETE  9 23 GDPKHPKSF  9 24GGQEKSAAG  9 25 GPCPGCPPC  9 26 PPCPGCPPC  9 27 DNEEKPTPEQD 11 28GNGGAPAQPKG 11 29 FEPPPATTTKSK 12 30 GNNDESNISFKEK 13 31 GDPKHPKSFTGWVA14 32 AQNPNKTNSNLDSSK 15 33 NKDNEAEPVTEGNAT 15 34 SKEGNGPDPDNAAKS 15 35GDKTPSTKSAGKVENK 16 36 GETDKEGKIIRIFDNSF 17 37 SSTSENNGNGNGNGGTD 17 38GNNDESNISFKEKSEEEE 18 39 GNNDESNISFKEKSKKKK 18 40 GNNDESNISFKEKSPPPP 1841 SSNKSTTGSGETTTAAGT 18 42 CGNNDESNISFKEKSKKKK 19 43GSPLSFESSVQLIVSDNSS 19 44 SNYAKKVVKQKNHVYTPVY 19 45ADVIAKIVEIVKGLIDQFTQK 21 46 GAASSLTYESSVQLVVSDNSS 21 47GGEPAAQAPAETPAAAAEAAS 21 48 GQTDNNSSQSQQPGSGTTNT 21 49SGALAATSDDDVKKAATVAIVA 22 50 SIVSTIIEVVKTIVDIVKKFKK 22 51SSGGGGVAADIGAGLADALTAP 22 52

The first four to five amino acids of the peptide moiety of alipopeptide may be selected from those listed for each position in Table2. This table is based upon Spohn et al., Vaccine, 22(19):2494-9, 2004;and Reutter et al., J. Peptide Res., 65, 375-383, 2005.

TABLE 2 1 2 3 4 5 D D A D D E E D E E F G E H H G K G N K K P H R M Q QM S N R R R T R S S S S T T

The lipopeptide may be an RR- or RS-stereoisomer, or mixture thereof,with respect to the stereochemistry of the N-terminal lipoamino acid.The lipopeptide may be water-soluble.

3. Treatment of Stress

An agent that induces NF-κB activity may be used to protect normal cellsfrom conditions or treatments that cause cellular stress, therebytriggering apoptosis. Representative examples of conditions ortreatments include cancer treatments, e.g., radiation therapy orchemotherapy; temperature shock; exposure to harmful doses of radiation,e.g., workers in nuclear power plants, the defense industry orradiopharmaceutical production, or soldiers; cell aging; wounding;poisoning; and infection.

The agent may be administered simultaneously or metronomically withother treatments. The term “simultaneous” or “simultaneously” as usedherein, means that the agent and other treatment be administered within48 hours, preferably 24 hours, more preferably 12 hours, yet morepreferably 6 hours, and most preferably 3 hours or less, of each other.The term “metronomically” as used herein means the administration of theagent at times different from the other treatment and at a certainfrequency relative to repeat administration.

The agent may be administered at any point prior to exposure to thestress including, but not limited to, about 48 hr, 46 hr, 44 hr, 42 hr,40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22 hr, 20hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 fir, 3 hr, 2 hr, or1 hr prior to exposure. The agent may be administered at any point afterexposure to the stress including, but not limited to, about 1 hr, 2 hr,3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20 hr, 22 hr,24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40 hr, 42 hr, 44hr, 46 hr, or 48 hr after exposure.

a. Constitutively Active NF-κB Cancer

The condition may be a constitutively active NF-κB cancer. The agentthat induces NF-κB activity may be administered in combination with acancer treatment, such as chemotherapy or radiation therapy.

The cancer treatment may comprise administration of a cytotoxic agent orcytostatic agent, or combination thereof. Cytotoxic agents preventcancer cells from multiplying by: (1) interfering with the cell'sability to replicate DNA and (2) inducing cell death and/or apoptosis inthe cancer cells. Cytostatic agents act via modulating, interfering orinhibiting the processes of cellular signal transduction that regulatecell proliferation.

Classes of compounds that may be used as cytotoxic agents include thefollowing: alkylating agents (including, without limitation, nitrogenmustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas andtriazenes): uracil mustard, chlormethine, cyclophosphamide (Cytoxan®),ifosfamide, melphalan, chlorambucil, pipobroman, triethylene-melamine,triethylenethiophosphoramine, busulfan, carmustine, lomustine,streptozocin, dacarbazine, and temozolomide; antimetabolites (including,without limitation, folic acid antagonists, pyrimidine analogs, purineanalogs and adenosine deaminase inhibitors): methotrexate,5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine,6-thioguanine, fludarabine phosphate, pentostatine, and gemcitabine;natural products and their derivatives (for example, vinca alkaloids,antitumor antibiotics, enzymes, lymphokines and epipodophyllotoxins):vinblastine, vincristine, vindesine, bleomycin, dactinomycin,daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c, paclitaxel(paclitaxel is commercially available as Taxol®), mithramycin,deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons (preferablyIFN-α), etoposide, and teniposide.

Other proliferative cytotoxic agents are navelbene, CPT-11, anastrazole,letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, anddroloxafine.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents that may be used include, but are not limited to,allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC608832), thiocolchicine NSC 361792), trityl cysteine (NSC 83265),vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574),natural and synthetic epothilones including but not limited toepothilone A, epothilone B, and discodermolide (see Service, (1996)Science, 274:2009) estramustine, nocodazole, MAP4, and the like.Examples of such agents are also described in Bulinski (1997) J. CellSci. 110:3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou(1997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985;and Panda (1996) J. Biol. Chem 271:29807-29812.

Also suitable are cytotoxic agents such as epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes such as cis-platin andcarboplatin; biological response modifiers; growth inhibitors;antihormonal therapeutic agents; leucovorin; tegafur; and haematopoieticgrowth factors.

Cytostatic agents that may be used include, but are not limited to,hormones and steroids (including synthetic analogs): 17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone,fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, and zoladex.

Other cytostatic agents are antiangiogenics, such as matrixmetalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU6668 arealso included. Anti-Her2 antibodies from Genentech may also be utilized.A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Alsoincluded are Imclone antibody C225 immunospecific for the EGFR, and srcinhibitors.

Also suitable for use as a cytostatic agent is Casodex® (bicalutamide,Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen Tamoxifen® which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include epidermal growth factor inhibitors, Her-2 inhibitors,MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Srckinase inhibitors, and PDGF inhibitors.

The cancer treatment may comprise radiation therapy. The radiationtherapy may be external beam radiation, internal radiation therapy, orconformal radiation therapy, in which a computer is used to shape thebeam of radiation to match the shape of the tumor. The radiation used inradiation therapy may come from a variety of sources, including anx-ray, electron beam, or gamma rays. The doses and timing ofadministration of the radiation during radiation therapy can and willvary depending on the location and extent of the cancer. The agent thatinduces NF-κB activity may be administered with a radioprotective agent(see section 3d) in combination with the radiation therapy, as describedabove.

Cancers that may be treated include, but are not limited to, thefollowing: carcinoma including that of the bladder (includingaccelerated and metastatic bladder cancer), breast, colon (includingcolorectal cancer), kidney, liver, lung (including small and non-smallcell lung cancer and lung adenocarcinoma), ovary, prostate, testes,genitourinary tract, lymphatic system, larynx, pancreas (includingexocrine pancreatic carcinoma), mouth, pharynx, esophagus, stomach,small intestine, colon, rectum, gall bladder, cervix, thyroid, and skin(including squamous cell carcinoma); hematopoietic tumors of lymphoidlineage including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocyticlymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineageincluding acute and chronic myelogenous leukemias, myelodysplasticsyndrome, myeloid leukemia, and promyelocytic leukemia; tumors of thecentral and peripheral nervous system including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal originincluding fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and othertumors including melanoma, xenoderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer, and teratocarcinoma.

b. Treatment of Side Effects from Cancer Treatment

The condition may also be damage to normal tissue attributable to thetreatment of a constitutively active NF-κB cancer. The agent thatinduces NF-κB activity may be administered in combination with a cancertreatment as described above.

c. Modulation of Cell Aging

The condition may also be cell aging.

d. Radiation

The condition may also be exposure to radiation. Exposure to ionizingradiation (IR) may be short- or long-term, it may be applied as a singledose or multiple doses, to the whole body or locally. Thus, nuclearaccidents or military attacks may involve exposure to a single high doseof whole body irradiation (sometimes followed by a long-term poisoningwith radioactive isotopes). Likewise, a single dose of radiation isgenerally used for the pretreatment of bone marrow transplant patientswhen it is necessary to prepare the host's hematopoietic organs for thedonor's bone marrow by “cleaning” them from the host blood precursors.

At the molecular and cellular level, radiation particles may lead tobreakage in the DNA and cross-linking between DNA, proteins, cellmembranes and other macromolecular structures. Ionizing radiation mayalso induce secondary damage to the cellular components by giving riseto free radicals and reactive oxygen species (ROS). Multiple repairsystems counteract this damage, such as several DNA repair pathways thatrestore the integrity and fidelity of the DNA, and antioxidant chemicalsand enzymes that scavenge the free radicals and ROS and reduce theoxidized proteins and lipids. Cellular checkpoint systems are present todetect the DNA defects and delay cell cycle progression until the damageis repaired or a decision to commit the cell to growth arrest orprogrammed cell death (apoptosis) is reached.

At the organism level, the immediate effects of low and moderate levelsof radiation are largely caused by cell death, which leads toradiation-induced inflammation. At higher radiation levels, theso-called hematopoietic and gastrointestinal syndromes lead toshort-term radiation-induced death. The hematopoietic syndrome ischaracterized by the loss of hematopoietic cells and their progenitors,thereby making it impossible to regenerate blood and the lymphoidsystem. Death usually occurs as a consequence of infection (due toimmunosuppression), hemorrhage and/or anemia. The gastrointestinalsyndrome is characterized by massive cell death in the intestinalepithelium, predominantly in the small intestine, followed by thedisintegration of the intestinal wall and death from bacteriemia andsepsis. The hematopoietic syndrome manifests itself at lower doses ofradiation and leads to a more delayed death than the gastrointestinalsyndrome. Very high doses of radiation can cause nearly instant death byeliciting neuronal degeneration.

Organisms that survive a period of acute toxicity of radiation maysuffer long-term consequences that include radiation-inducedcarcinogenesis and fibrosis that develop in exposed organs (e.g.,kidney, liver or lungs) months and even years after irradiation.

Inducers of NF-κB possess strong pro-survival activity at the cellularlevel and may be used to treat the effects of natural radiation events,exposure to low doses of radiation, radiation administered as part ofcancer therapy, or nuclear accidents. Moreover, since inducers of NF-κBacts through mechanisms different from all presently knownradioprotectants, they may be used in combination with otherradioprotectants, thereby, dramatically increasing the scale ofprotection from ionizing radiation.

Historically, radioprotectants have generally been antioxidants and freeradical scavengers—both synthetic and natural. More recently, cytokinesand growth factors have been added to the list of radioprotectants; themechanism of their radioprotection is considered to be due to theirfacilitating effect on the regeneration of sensitive tissues. There isno clear functional distinction between the two groups ofradioprotectants, however, since some cytokines induce the expression ofthe cellular antioxidant proteins, such as manganese superoxidedismutase (MnSOD) and metallothionein, their use may be advantageous.

The radioprotectants may be any agent that treats the effects ofradiation exposure including, but not limited to, antioxidants, freeradical scavengers, cytokines, flagellin and latent TGFβ. Antioxidantsand free radical scavengers that may be used include, but are notlimited to, thiols, such as cysteine, cysteamine, glutathione andbilirubin; amifostine (WR-2721); vitamin A; vitamin C; vitamin E; andflavonoids such as Indian holy basil (Ocimum sanctum), orientin andvicenin. Cytokines and growth factors confer radioprotection byreplenishing and/or protecting the radiosensitive stem cell populations.Cytokines that may be used include stem cell factor (SCF, c-kit ligand),Flt-3 ligand, interleukin-1 fragment IL-1b-rd, and keratinocyte growthfactor (KGF). Several other factors, while not cytokines by nature,stimulate the proliferation of the immunocytes, and thus, may be used.These include, 5-AED (5-androstenediol), which is a steroid thatstimulates the expression of cytokines, and synthetic compounds, such asammonium tri-chloro(dioxoethylene-O,O′-)tellurate (AS-101). Latent TGFβ,flagellin and flagellin derivatives are strong inducers of NF-κBactivity as shown in International Patent Application Nos.PCT/US2004/040656 and PCT/US2004/040753, and U.S. Patent Application No.60/693,826, the contents of which are incorporated herein by reference.

4. Composition

Provided herein also are compositions comprising a therapeuticallyeffective amount of an inducer of NF-κB. The composition may be apharmaceutical composition, which may be produced using methods wellknown in the art. As described above, the composition comprising aninducer of NF-κB may be administered to a mammal for the treatment ofconditions associated with apoptosis including, but not limited to,exposure to radiation, side effect from cancer treatments, stress andcell aging. The composition may also comprise additional agentsincluding, but not limited to, a radioprotectant or a chemotherapeuticdrug.

a. Administration

Compositions provided herein may be administered in any mannerincluding, but not limited to, orally, parenterally, sublingually,transdermally, rectally, transmucosally, topically, via inhalation, viabuccal administration, or combinations thereof. Parenteraladministration includes, but is not limited to, intravenous,intraarterial, intraperitoneal, subcutaneous, intramuscular,intrathecal, and intraarticular. For veterinary use, the composition maybe administered as a suitably acceptable formulation in accordance withnormal veterinary practice. The veterinarian can readily determine thedosing regimen and route of administration that is most appropriate fora particular animal.

b. Formulation

Compositions provided herein may be in the form of tablets or lozengesformulated in a conventional manner. For example, tablets and capsulesfor oral administration may contain conventional excipients including,but not limited to, binding agents, fillers, lubricants, disintegrantsand wetting agents. Binding agents include, but are not limited to,syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch andpolyvinylpyrrolidone. Fillers include, but are not limited to, lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate. Wetting agents include, but are not limited to,sodium lauryl sulfate. Tablets may be coated according to methods wellknown in the art.

Compositions provided herein may also be liquid formulations including,but not limited to, aqueous or oily suspensions, solutions, emulsions,syrups, and elixirs. The compositions may also be formulated as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may contain additives including, but notlimited to, suspending agents, emulsifying agents, nonaqueous vehiclesand preservatives. Suspending agent include, but are not limited to,sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin,hydroxyethylcellulose, carboxymethyl cellulose, aluminum stearate gel,and hydrogenated edible fats. Emulsifying agents include, but are notlimited to, lecithin, sorbitan monooleate, and acacia. Nonaqueousvehicles include, but are not limited to, edible oils, almond oil,fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions provided herein may also be formulated as suppositories,which may contain suppository bases including, but not limited to, cocoabutter or glycerides. Compositions provided herein may also beformulated for inhalation, which may be in a form including, but notlimited to, a solution, suspension, or emulsion that may be administeredas a dry powder or in the form of an aerosol using a propellant, such asdichlorodifluoromethane or trichlorofluoromethane. Compositions providedherein may also be formulated as transdermal formulations comprisingaqueous or nonaqueous vehicles including, but not limited to, creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

Compositions provided herein may also be formulated for parenteraladministration including, but not limited to, by injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The composition may also be providedin a powder form for reconstitution with a suitable vehicle including,but not limited to, sterile, pyrogen-free water.

Compositions provided herein may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The compositions may be formulated withsuitable polymeric or hydrophobic materials (as an emulsion in anacceptable oil, for example), ion exchange resins, or as sparinglysoluble derivatives (as a sparingly soluble salt, for example).

c. Dosage

A therapeutically effective amount of the agent required for use intherapy varies with the nature of the condition being treated, thelength of time that induction of NF-κB activity is desired, and the ageand the condition of the patient, and is ultimately determined by theattendant physician. In general, however, doses employed for adult humantreatment typically are in the range of 0.001 mg/kg to about 200 mg/kgper day. The dose may be about 1 μg/kg to about 100 μg/kg per day. Thedesired dose may be conveniently administered in a single dose, or asmultiple doses administered at appropriate intervals, for example astwo, three, four or more sub-doses per day. Multiple doses often aredesired, or required, because NF-κB activity in normal cells may bedecreased once the agent is no longer administered.

The dosage of an inducer of NF-κB may be at any dosage including, butnot limited to, about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg,125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg,450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg,775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925μg/kg, 950 μg/kg, 975 μg/kg or 1 mg/kg.

5. Screening Methods

The method provided herein also relates to methods of identifying agentsthat induce NF-κB activity. An agent that induces NF-κB activity may beidentified by a method comprising adding a suspected inducer of NF-κBactivity to an NF-κB activated expression system, comparing the level ofNF-κB activated expression to a control, whereby an inducer of NF-κBactivity is identified by the ability to increase the level of NF-κBactivated expression system.

Candidate agents may be present within a library (i.e., a collection ofcompounds). Such agents may, for example, be encoded by DNA moleculeswithin an expression library. Candidate agents may be present inconditioned media or in cell extracts. Other such agents includecompounds known in the art as “small molecules,” which have molecularweights less than 10⁵ daltons, preferably less than 10⁴ daltons, andstill more preferably less than 10³ daltons. Such candidate agents maybe provided as members of a combinatorial library, which includessynthetic agents (e.g., peptides) prepared according to multiplepredetermined chemical reactions. Those having ordinary skill in the artwill appreciate that a diverse assortment of such libraries may beprepared according to established procedures, and members of a libraryof candidate agents can be simultaneously or sequentially screened asdescribed herein.

The screening methods may be performed in a variety of formats,including in vitro, cell-based and in vivo assays. Any cells may be usedwith cell-based assays. Preferably, cells that may be used includemammalian cells, more preferably human and non-human primate cells.Cell-base screening may be performed using genetically modified tumorcells expressing surrogate markers for activation of NF-κB. Such markersinclude, but are not limited to, bacterial β-galactosidase, luciferaseand enhanced green fluorescent protein (EGFP). The amount of expressionof the surrogate marker may be measured using techniques standard in theart including, but not limited to, colorimetery, luminometery andfluorimetery.

The conditions under which a suspected modulator is added to a cell,such as by mixing, are conditions in which the cell can undergoapoptosis or signaling if essentially no other regulatory compounds arepresent that would interfere with apoptosis or signaling. Effectiveconditions include, but are not limited to, appropriate medium,temperature, pH and oxygen conditions that permit cell growth. Anappropriate medium is typically a solid or liquid medium comprisinggrowth factors and assimilable carbon, nitrogen and phosphate sources,as well as appropriate salts, minerals, metals and other nutrients, suchas vitamins, and includes an effective medium in which the cell can becultured such that the cell can exhibit apoptosis or signaling. Forexample, for a mammalian cell, the media may comprise Dulbecco'smodified Eagle's medium containing 10% fetal calf serum.

Cells may be cultured in a variety of containers including, but notlimited to tissue culture flasks, test tubes, microtiter dishes, andpetri plates. Culturing is carried out at a temperature, pH and carbondioxide content appropriate for the cell. Such culturing conditions arealso within the skill in the art.

Methods for adding a suspected modulator to the cell includeelectroporation, microinjection, cellular expression (i.e., using anexpression system including naked nucleic acid molecules, recombinantvirus, retrovirus expression vectors and adenovirus expression), use ofion pairing agents and use of detergents for cell permeabilization.

This invention has multiple aspects, illustrated by the followingnon-limiting examples.

EXAMPLE 1 p53 Deficiency Accelerates Development of the GI Syndrome inMice

The primary cause of death from ionizing radiation (IR) of mammalsdepends on the radiation dose. At doses of up to 9-10 Gy, mice die 12-20days later, primarily from lethal bone marrow depletion, i.e., thehematopoietic (HP) syndrome. At this dose, irradiated mice can berescued from lethality by bone marrow transplantation. Animals thatreceived >13-15 Gy die between 7-12 days after treatment (beforehematopoietic syndrome could kill them) from complications of damage tothe small intestine, i.e., the gastrointestinal (GI) syndrome. It iswell known that cell proliferation of the epithelial cells of the smallintestine is limited to the crypts where stem cells and earlyproliferating progenitors are located. After a couple of cell divisions,already differentiated descendants of crypt stem cells move up the villito be shed at the villar tip. In the small intestine of the mouse, theentire “trip” of the cell (i.e., from the proliferative compartment tothe tip of the villus) normally takes between 3 and 5 days. Althoughreaction of the small intestine to gamma radiation has been wellexamined at a pathomorphological level, the exact cause of GI lethality,including the primary event, still remains unclear. Death may occur as adirect consequence of the damage to the epithelial crypt cells, followedby denudation of the villi leading to fluid and electrolyte imbalance,bacteremia, and endotoxemia. In addition to inflammation and stromalresponses, endothelial dysfunctions appear to be important factorscontributing to lethality.

In both the HP and GI syndromes, lethal tissue damage results frommassive p53 dependent apoptosis. Furthermore, it has been shown that p53dependent hair loss (alopecia) occurs as a result of experimentalchemotherapy or radiation. Thus, it appears that p53 could play a rolein sensitizing cells to genotoxic stress.

To examine the role of p53 in radiation-induced death, mice were treatedwith the small molecule inhibitor of p53, pifithrin-alpha (PFTα)(Komarov et al., Science 285:1733-7, 1999) immediately prior to gammairradiation. C57Bl/6J mice (6-8 weeks old males were used here andbelow, if not indicated otherwise) were injected intraperitoneally with10 mg/kg of PFTα and then irradiated using a Shepherd 4000 Ci ¹³⁷Cesiumsource at a dose rate of 4 Gy per minute. PFTα protected mice from asingle 9 Gy dose of gamma radiation or a fractioned cumulative radiationdose of 12.5 Gy (5×2.5 Gy). In contrast, PFTα had no effect on thesurvival of mice treated with single high doses, i.e., 12.5 or 25 Gy, ofIR (FIG. 1a ).

To further examine the role of p53 in the GI syndrome, wild type andp53-deficient mice were exposed to low (10 Gy) and high (15 Gy) doses ofgamma radiation. As shown in FIG. 1b , p53-deficient mice were resistantto low doses of radiation that kill through the HP syndrome, but muchmore sensitive to higher doses of radiation that kill through the GIsyndrome. Haematoxylin-eosin stained paraffin sections of the smallintestinal from wild type and p53-null mice at 0, 24, 48, 72, and 96 hrafter a 15 Gy dose of gamma radiation are shown in FIG. 1d . Thep53-deficient mice exhibited accelerated epithelial cell damage. TUNELstaining in the crypts (at 24 hr) revealed that apoptosis was evident inwild type but not in p53-deficient epithelium. To examine this further,wild-type mice were exposed to 11 Gy of total body irradiation and then,12 hr later, injected with 1.5×10⁷ bone marrow cells from wild type orp53-null syngeneic C57Bl/6J mice. (This dose of radiation caused 100%lethality in nonreconstituted control mice). Two months later, aftercomplete recovery of hematopoiesis, the two groups of animals weretreated with 15 Gy of total body gamma radiation. As shown in FIG. 1c ,there was no difference in death rates between the two groups mice thatdiffered in the p53 status of their bone marrow (both had wild-typeintestinal cells).

The dynamics of cell proliferation and cell survival were furtherexamined in the small intestines of wild type and p53-null mice.Four-week old wild type and p53-null mice were injectedintraperitoneally with ¹⁴C-thymidine (10 μCi per animal) and then halfof each group was exposed to 15 Gy of gamma radiation. Autoradiographsof whole-body sections revealed that after 24 hrs, the cells in theintestinal crypts of p53-deficient mice continued to proliferate,whereas those in the wild type mice were quiescent (FIG. 2a , left).Four-week old wild type and p53-null mice were treated with 15 Gy ofgamma radiation, and 2 hr before being sacrificed they were injectedwith BrdU (50 mg/kg) and the intestines were immunostained. At 24 hrafter irradiation, there were many proliferating cells in the p53-nullmice, but few in the wild type mice. In contrast, at 96 hr, there werefew proliferating cells in the p53-null mice, whereas the wild type micedisplayed more labeled cells.

To characterize this further, the number of BrdU positive cells werecounted in the small intestines of wild-type and p53-null mice atdifferent time points after 15 Gy of gamma radiation. Three animals wereanalyzed for each time point, five ilial cross sections were preparedfrom each animal and analyzed microscopically to estimate the number ofcrypts and villi. Numbers of BrdU-positive cells in the crypts werecounted in five random fields under 200× magnification (100-300 crypts)and the average number of BrdU-positive cells was plotted (FIG. 2b ).The number of BrdU-positive cells in the p53-null mice peaked at 10 hrand then declined, whereas the number of BrdU-positive cells in wildtype mice declined during the first 20 hrs and then increased. Thelocation of BrdU-labeled cells was traced in the small intestines ofwild type and p53-null mice at different time points after 15 Gy ofgamma radiation. BrdU was injected 30 min before irradiation and micewere sacrificed at 0, 48, 72, and 96 hr. In p53-null mice, there was anaccelerated migration of BrdU-labeled cells from the crypts to the villi(compare wild type and p53-null at 48 hr), followed by rapid eliminationof the labeled cells in p53-null mice (FIG. 2c ).

Thus, continuous cell proliferation in the crypts of irradiatedp53-deficient epithelium correlates with the accelerated death ofdamaged cells of the crypt and rapid destruction of the villi. In wildtype mice, however, p53 prolongs survival by inducing growth arrest inthe crypts of the small intestine, thereby preserving integrity of theintestine. Thus, the proapoptotic function of p53 promotes thehematopoietic syndrome, while its growth arrest function delaysdevelopment of the gastrointestinal syndrome. Thus, pharmacologicalsuppression of p53 would be useless (if not detrimental) against the GIsyndrome. Therefore, it is necessary to develop alternative approachesto radioprotection of epithelium of small intestine that will rely onanother mechanism, such as, for example, activation of NF-κB andsubsequent inhibition of cell death.

EXAMPLE 2 Lipopeptides Delay Mouse Death Caused by Total BodyGamma-irradiation

Lipopeptides are potent activators of NF-κB and, as such, may act asinhibitors of apoptotic death. To determine whether lipopeptidesfunction as radioprotectants, various lipopeptides were initially testedto determine the maximal tolerable dose (MTD). Various lipopeptides werethen tested to measure their protective effect in NIH Swiss mice tolethal hematopoietic or nd gastrointestinal syndromes after exposure to10 Gy or 13-15 Gy of total body gamma radiation, respectively.Lipopeptides (0.3-10 μg/mouse) were administered subcutaneously 30minutes prior to irradiation. Lipopeptides tested that providedradioprotection are set forth in Table 3.

TABLE 3 SEQ  Peptide  Peptide Sequence ID NO Length N-acylation SKKKK 85 R-Pam2 SKKKK 8 5 R-Pam3 FEPPPATTT 22 9 Pam2 GNNDESNISFKEK 31 13 Pam2GDPKHPKSF 24 9 Pam2 GETDKEGKIIRIFDNSF 37 17 Pam2

The results of a representative experiment using R-PAM₂C-SKKKK (SEQ IDNO: 8) (hereafter, this compound is called, CBLB601) at <0.1 MTD areshown in FIG. 3. As expected, control mice irradiated with 10 Gy diedbetween 11 and 15 days post-treatment, while all animals that receivedCBLB601 lived beyond 35 days post-treatment. Similarly, control miceirradiated with 13 Gy died between 6 and 10 days post-treatment, whileall but one animal that received CBLB601 lived beyond 35 dayspost-treatment. The radioprotective ability of CBLB601 was furtheranalyzed by measuring the effect of radiation on spleen size. As shownin FIG. 4, mice treated with CBLB601 showed significantly less reductionin the size of the spleen. CBLB601 also protected the thymus fromradiation (data not shown). The ability of CBLB601 to effectivelyprotect splenocytes, support fast recovery of the thymus, and protectthe GI tract from radiation damage indicates that lipopeptides may beused as radioprotectants.

EXAMPLE 3 Radioprotective Efficacy of a Single Dose of CBLB601 againstTotal Body Gamma-irradiation

CBLB601, a R-Pam₂-lipopeptide with the peptide moiety consisting ofC-SKKKK (SEQ ID NO: 8), was selected for more detailed characterizationas a radioprotector based upon its ability to activate NF-κB andpreliminary in vivo data on radioprotection in NIH-SWISS mice (seeExample 2). The objectives of this study were to determine the optimaldose, route of administration, and time of administration of CBLB601 toserve as a protector. ICR female mice of 10-15 weeks of age were used,with 10-15 animals per group or condition.

The dose of NOAEL (No Obvious Adverse Effects Level) was determined byinjecting intraperitoneally (i.p) ICR mice with the increasing doses ofCBLB601 (0.3, 1, 3, 10, 30, 60, 100 μg/mouse). Control mice wereinjected with PBS. The mice were observed for two weeks. During thefirst week they were weighed daily. There were no differences in weightbetween the CBLB601-treated and control mice. Mortality was observed 1-2days post-treatment at the 100 μg of CBLB601/mouse, but not at any ofthe lower doses. However, at 60 μg dose, the mice showed signs ofmorbidity, such as slow motion and scruffy fur, around 3-4 days aftertreatment. At the 30 μg dose, there were no noticeable differencesbetween the treated and control mice. Thus, NOAEL for CBLB601 wasdetermined to be 30 μg/mouse.

The optimal intraperitoneal administration schedule of CBLB601 wasdetermined by injecting the compound at different times prior toirradiation. Previously, it was found that CBLB601 was protectiveagainst 10 Gy but not higher irradiation doses (see Example 2).Therefore, all of the optimization experiments were performed with 10 Gyof total body irradiation (TBI). A dose of 3 μg of BCLB601/mouse (1/10NOAEL) was chosen as the starting dose. Thus, 3 μg of CBLB601 wasinjected i.p. into ICR mice at 0.5 h, 1 h, 6 h, and 24 h or 1 h, 24 h,48 h, 72 h, 96 h prior to 10 Gy of TBI (as described in Example 1).Following irradiation, the mice were observed for 30 days and theirsurvival was recorded. The results of these experiments are summarizedin FIG. 5. Injection of CBLB601 24 hrs before irradiation clearlyyielded the best radioprotection (90-100% 30-day survival). When thecompound was administered 48 hrs before irradiation, the radioprotectionwas ˜30%. Administration of CBLB601 at 1 hr prior to irradiationproduced inconsistent results ranging from 80% protection in oneexperiment (FIG. 5B) to almost no protection (20%) in another experiment(FIG. 5A). No radioprotection was observed when the drug wasadministered at 0.5 h (10%), 6 h (20%), 72 h, and 96 h prior to TBI.

To determine the optimal radioprotective dose of CBLB601, the timing ofinjection and level of irradiation were kept constant, while the dose ofCBLB601 was varied. ICR mice were injected i.p. with 1, 3, 10, 20, or 30μg of CBLB601/mouse or 0.1, 0.3, 1, 3, 10, or 15 of CBLB601/mouse 24 hrsprior to irradiation (10 Gy of TBI), and their survival was monitoredfor 30 days. The best protective dose was 3 μg/mouse, which supported asurvival of 100% (FIGS. 6A and B). Almost similar efficacy was reachedwith the 1 and 10 μg/mouse doses, which rescued 90% of the mice (in oneexperiment). In contrast, higher doses of CBLB601 (20 and 30 μg/mouse)led to accelerated mortality when administered in combination withirradiation, as compared to PBS injected control mice. Some signs ofthis combined toxicity were detectable already at the 10 μg/mouse dose.Thus, the optimal protective dose of CBLB601 was determined to be 3μg/mouse. Thus, while it appears that CBLB601 conferred radioprotectionat doses 10-30-fold lower than the NOAEL, the margins of safety ofCBLB601 were significantly reduced when CBLB601 was administered incombination with irradiation.

To determine the level of radiation that was protected by CBLB601,radioprotectant-treated and control mice were exposed to increasinglevels of TBI. Groups of ICR mice were injected i.p. with either 3 μgCBLB601/mouse or PBS, and then 24 h later they received 10, 11, 12, 13,14, and 15 Gy doses of TBI. Survival was recorded over 30 days. FIG. 7Ashows the radiation dose-dependent mortality of mice injected with PBS.All of the mice irradiated with 10-11 Gy of TBI died between days 12-14post irradiation, which was typical for death due to hematopoieticfailure. All of the mice that received 14-15 Gy of TBI died between days7-9 post irradiation, which was typical for mortality due toradiation-induced intestinal damage. Mice that were irradiated with12-13 Gy of TBI died at intermediate times, which was typical for amixed etiology of radiation-induced mortality. FIG. 7B shows theradiation dose-dependent mortality of mice pretreated with CBLB601. Miceinjected with 3 μg of CBLB601 24 hrs prior to irradiation were, asexpected, fully protected from 10 Gy of TBI. Despite the obviousdifferences in survival between the control and treated mice, theprotective effects of CBLB601 did not reach statistical significanceunder this setting. To reach a statistically significant 10% differencebetween control and CBLB601-treated mice, experimental groups of atleast 50 mice must be used. Although CBLB601 rescued 100% of mice from10 Gy of TBI, its protection at 11 Gy of TBI was only 20%. There was noprotection from irradiation at levels higher than 11 Gy, indicating thatCBLB601 was unable to rescue animals from the gastrointestinal componentof radiotoxicity. Moreover, at irradiation doses of 11, 12 and 13 Gy,the CBLB601-treated mice died with an accelerated kinetics, i.e.,similar to those that received 14-15 Gy doses, as compared toPBS-injected control mice. This may be indicative of a combined toxicityof the drug and the irradiation. Thus, it appears that at higherirradiation levels, CBLB601 did not confer protection and it was alsomore toxic.

CBLB601 was administered intramuscularly to determine the optimal routeof administration of this compound (especially since this would be thepreferred route of administration for humans). FIG. 8 shows the survivalrates of mice injected intramuscularly (i.m.) with 1, 3, or 10 μg ofCBLB601/mouse 24 hr before irradiation. All three doses of CBLB601imparted radioprotection; and there was no sign of combined toxicity atthe 10 μg/mouse i.m. dose, as there was for the 10 μg/mouse i.p. dose(FIG. 6). In another experiment, increasing doses of CBLB601administered i.m. were tested against 10, 11, and 12 Gy doses of HBI(FIG. 9). There was a non-statistically significant shift to increasedmortality of mice injected with the higher doses (10, 30 μg) of CBLB601prior to exposure to 10 Gy of TBI. A summary of the dose-dependency ofthe radioprotection of CBLB610 administered i.p. or i.m. 24 h prior to10 Gy of TBI is shown in FIG. 10. From these data, it was concluded thatthe i.m. route of administration was as effective as the i.p. route, andthe optimal dose (3 μg/mouse) was the same for both routes ofadministration. Moreover, it was concluded that i.m. delivery may besafer because it had a higher therapeutic index (toxic dose/effectivedose): 10-30 for i.m. administration (FIGS. 8 and 9: 30 μg/mouse/1-2μg/mouse) vs. 3-10 for i.p. administration (FIG. 6: 10 μg/mouse/1-3μg/mouse). Additionally, recalculation of the effective dose per bodyweight revealed a smaller window for intraperitoneal delivery comparedto intramuscular delivery; i.e., 90-110 μg/kg for i.p. delivery and60-115 μg/kg for i.m. delivery (FIG. 11).

The optimal schedule of intramuscular administration of CBLB601 wasdetermined by varying the time between drug delivery and irradiation.ICR mice were injected i.m. with 3 μg of CBLB601/mouse 24 h, 6 h, 3 h,and 1 h prior to TBI and +1 h, +3 h after TBI (FIG. 12A), as well as 48h, 36 h, 24 h, 12 h, and 6 h prior to TBI (FIG. 12B). These experimentsrevealed that, similar to the intraperitoneal route of delivery, theoptimal time for intramuscular delivery of CBLB601 was 24 h prior to 10Gy of TBI. Intramuscular administration of 3 μg of CBLB610/mouse after(1 h and 3 h) 10 Gy of TBI had no protective effect (FIG. 12A).Moreover, higher doses of CBLB601 (10 and 30 μg/mouse) injected i.m. 1 hafter 10 Gy of TBI caused increased levels of combined toxicity (FIG.13C). These data are summarized in FIG. 13.

To determine DMF (dose modification factor), 3 μg of CBLB601/mouse wasinjected i.m. 24 hr prior to irradiation and control mice were injectedwith PBS. Both drug-injected and control groups received a single doseof TBI covering the dose range that leads to 10-90% mortality within thechosen time intervals (7 days for the gastrointestinal syndromemortality, and 30 days for the hematopoietic syndrome mortality). Thepercent mortality-radiation dose graphs were built and LD_(50/7) andLD_(50/30) (LD₅₀ at 7 days and 30 days, respectively) were calculatedusing probit or logit statistical analysis. DMF (also known as dosereduction factor, DRF) was calculated as a ratio of radiation LD₅₀ forCBLB601-treated groups and radiation LD₅₀ for vehicle-treated groups ofmice for the chosen survival time point, 7 or 30 days. To calculateDMF₃₀, which is the DMF at day 30 post-irradiation, the radiationLD_(50/30) values for mice treated with PBS or 3 μg of CBLB601 wasdetermined. For this, PBS-injected groups were irradiated with doses of6, 6.5, 7, and 7.7 Gy of TBI, and CBLB601-injected groups wereirradiated with doses of 10, 10.5, 11, 11.5, and 12 Gy of TBI. TheLD_(50/30) for CBLB601 was calculated using ProBit analysis and wasestimated to be in the range of 11.07-11.61Gy, with the average of˜11.32 Gy (see FIG. 14). However, inconsistency in the response to someradiation doses (7 Gy appeared non-toxic whereas 6.5 Gy caused 60%lethality at 30 days) in the control mice precluded an accuratecalculation of the DMF₃₀ for CBLB601. Nevertheless, it was expected thatthe LD_(50/30) for ICR mice was around 7 Gy [an average for the majorityof mouse strains; Monobe et al., Radiother Oncology 73 Suppl 2: S12709,2004)]. Thus, the value of DMF₃₀ for CBLB601 was estimated roughly as˜1.6.

EXAMPLE 4 Immune Status of Mice Rescued from Lethal Doses of TBI byCBLB601

Mice that have been rescued from 10 Gy of TBI by administration ofCBLB601 may have compromised immune systems, and hence, may not have“normal” lives. To check the status of their immune systems, ICR micewere exposed to lethal (10 Gy) or non-lethal (6 Gy) doses of TBI 24 hrafter they had been injected (i.m.) with 3 μg CBLB601/mouse or PBS.Groups of five mice from each condition were sacrificed very three daysand their spleens and thymuses were removed and weighed. The weights ofthe spleens were normalized to the body weights of the correspondinganimals and the results are presented in FIG. 15. PBS-treated mice thatwere exposed to 6 Gy of radiation took about 13-14 days to restore theirspleens to normal weights, whereas CBLB601-treated mice exposed to 6 Gyof radiation had normal spleen weights by day 8. PBS-injected animalsdid not survive 10 Gy of TBI, whereas CBLB601-injected mice not onlysurvived the lethal dose of radiation, but also completely restoredtheir spleen weights by day 13-14 post irradiation. It took a longerperiod of time (˜30 days) to restore the thymuses to normal weights (notshown).

The spleens and thymuses from control and CBLB601-injected mice wereexamined microscopically for morphological changes at 3, 10 and 30 daysafter irradiation. At 3 days post-irradiation, the spleen and thymus of10 Gy whole body irradiated control and CBLB601-treated mice revealedirradiation-induced lesions, including moderately severe or severelymphoid depletion of the spleen and thymus, and severe red pulp atrophyof the spleen. At 10 days post-irradiation, CBLB601-treated animalsshowed an improvement over the control animals in recovery from splenicred pulp atrophy; all CBLB601-treated animals showed mild to moderatemultifocal extramedullary hematopoiesis (EMH) whereas none of thecontrols displayed EMH. There was no evidence of recovery of the splenicwhite pulp depletion in either group at day 10. Regenerative lymphoidhyperplasia in the thymus was evident in both groups at day 10, with theregeneration slightly more advanced in the control that in theCBLB601-treated mice. By day 30 post-irradiation, all animals had normalsplenic red pulp, most animals showed full or nearly full recovery oflymphoid elements, and all had essentially normal thymuses.

To test the immune response of mice rescued from a lethal dose ofgamma-irradiation (10 Gy of TBI) by CBLB601, several groups of such micewere subjected to immunization with a strong antigen, Salmonellaflagellin. The mice were first immunized at 8, 18, or 20 weeks afterirradiation, at which time they were 18, 25, and 33 weeks of age,respectively. Non-irradiated naïve mice at 29 weeks of age were used asa positive control for the immune response. The mice received a boost offlagellin one week after the first immunization, and another boost threeweeks after the 2^(nd) boost; anti-flagellin antibody titers weremeasured in mouse serum. To test the secondary immune response, the micewere bled again a month later to insure reduction of the antibodytiters. This was followed by a 3^(rd) boost of the antigen injection andmeasurement of anti-flagellin antibodies titers 10 days later. At thetime of the 3^(rd) boost, the irradiation mice were 38, 30, and 23 weeksold, and the control mice were 34 weeks old.

The immune response one month after the first immunization (three weeksafter the first boost) is shown in FIG. 16A. The CBLB601-treatedirradiated mice mounted a robust humoral immune response againstflagellin that was indistinguishable from that of the naïve controlmice. It also appears that the level of immune response was notdependent on the time that had passed after irradiation, since allgroups displayed a good response. While there was some individualvariation in the 20-week post-irradiation group, it is well known thatimmune response can be impaired in older animals. Antibody titers werechecked again one month after the first bleed, and they were stillelevated seven weeks after immunization (FIG. 16B). The secondary immuneresponse (FIG. 16C) was stronger than the first response (FIG. 16A).These data strongly indicate that CBLB601 not only rescued mice fromlethal TBI, but also allowed for full restoration of a functional immunesystem.

EXAMPLE 5 Activation of NF-κB and Radioprotection Provided by OtherLipopeptides

Additional lipopeptides were synthesized and tested for activation ofNF-κB and their ability to protect mice from lethal doses of radiation.The names of the compounds and their key constituents are shown in Table4. For some of the compounds, the corresponding free peptides were alsosynthesized and tested.

TABLE 4 Compound Peptide  SEQ  Name N-acylation Sequence ID NO CBLB602Pam2 GQHHH 12 CBLB603 Pain2 GQHHM 11 CBLB604 Pam2 GSHHM 14 CBLB605 Pam2SQMHH 15 CBLB606 R-Pam2 GDPKHPKSF 24 CBLB607 Pam2 GDPKHPKSFTGWVA 32CBLB608 Pam2 FEPPPATTTKSK 30 CBLB611 Pam2 GETDKEGKIIRIFDNSF 37 CBLB612R-Pam2 VQGEESNDK 21 CBLB613 R-Pam2 GETDK 16 CBLB614 R-Pam2 QGEESNDK 20CBLB615 R-Pam2 GEESN 17 CBLB616 R-Pam2 TENVKE 19 CBLB617 R-Pam2 GEEDD 18

NF-κB-dependent reporter activity was measured in 293 cells thatexpressed the TLR2/YLR6 heterodimer. The in vitro activation of NF-κB byCBLB613 was comparable to that of CBLB601 (FIG. 17). The compoundsCBLB614 and CBLB615 have successively shorter derivatives of the peptideof CBLB612 (see Table 4). All three compounds activated the NF-κBreporter, and all were better activators than CBLB601 (FIG. 18A, B).None of the non-palmitoylated corresponding peptides activated the NF-κBreporter (FIG. 18B). The peptide component of CBLB617 has a (−4) charge,which should prevent it from interacting with negatively charged cellsurface markers. The NF-κB activation of CBLB617 was comparable to thatof CBLB601 (FIG. 19). Table 5 summarizes the in vitro activity andsolubility of all the compounds.

TABLE 5 Coumpound Solubility NF-κB activation CBLB601 Excellent 100CBLB602 Poor 0 CBLB603 Poor 0 CBLB604 Poor 0 CBLB605 Poor 2 CBLB606 Poor100 CBLB607 Poor 2 CBLB608 Excellent 2 CBLB611 Insoluble 0 CBLB612Excellent 200 CBLB613 Soluble 200 CBLB614 Soluble 200 CBLB615 Soluble200 CBLB616 Not tested Not tested CBLB617 Soluble 100

The in vivo radioprotective activity of some of the compounds was alsotested. ICR mice were injected intramuscularly with various doses of thetest compounds and then 24 hours later the mice were exposed to 10 Gy ofTBI. Survival was monitored for 30 days. FIG. 20 shows the protectiveactivity of CBLB613. Doses of 10, 30, and 82.5 μg of CBLB613/mouseprovided 100% protection, and were non-toxic in combination with theradiation (in contrast to CBLB601). The related compounds, CBLB612,CBLB614, and CBLB615 were tested for radioprotective activity against 10Gy of TBI. Doses of 1, 3, 10, and 30 μg/mouse (CBLB612 was also testedat 60 μg/mouse) were injected i.m. into ICR mice 24 hrs beforeirradiation. As shown in FIG. 21, all three of the compounds provided90-100% radioprotection when administered at the highest doses. Theradioprotection was clear dose-dependent, with no toxicity incombination with radiation. The potency of the compounds areCBLB612>CBLB614>CBLB615. The radioprotective activity of CBLB617 iscurrently under evaluation using the standard procedure. After 13 days,there was no visible toxicity at the highest dose (87.5 μg/mouse), whilethere was 7-90% survival at doses of 10-87.5 μg/mouse, respectively. Insummary, CBLB612 and CBLB613 are significantly better radioprotectorsthan CBLB601, and they have higher therapeutic indices (˜20 for CBLB612and CBLB613 vs. 3 for CBLB601).

The ability of CBLB612 to serve as a mitigator of lower dose radiationinjury was also examined. For this, 50 μg of CBLB612/mouse was injectedintramuscularly 1 hr after exposure to 8.5, 9, or 10 Gy of TBI.Treatment with CBLB612 increased the survival rate at every dose ofradiation (FIG. 22). For example, at 8.5 Gy, 90% of the CBLB612-treatedand 50% of the PBS-treated mice survived to 27 days (p=0.03), and, at 9Gy 70% of the CBLB612-treated and 50% of the PBS-treated mice survivedto 27 days (p=0.0006). No difference was observed betweenCBLB612-treated and control groups at lower radiation doses due to thelow control mice survival (not shown). These data indicate CBLB612 mayserve as a radiation mitigator, as well as a radioprotector.

The invention claimed is:
 1. A method for activating a Toll-likereceptor (TLR) in a cell, comprising contacting the cell with acomposition comprising a compound of the formula:

wherein, R₁ represents H or —CO—R₄; R₂, R₃, and R₄ independently are Hor optionally substituted C₆-C₂₀ aliphatic; X is a peptide comprising asequence selected from SEQ ID NOs: 17, 20, or 21; and Z is S or CH₂. 2.The method of claim 1, wherein the peptide comprises the sequence of SEQID NO:
 21. 3. The method of claim 1, wherein R₂, R₃, and R₄independently are H or optionally substituted C₈-C₁₆ aliphatic.
 4. Themethod of claim 1, wherein the compound is an RR or RS stereoisomer, ora mixture thereof.
 5. The method of claim 1, wherein the TLR is TLR2. 6.The method of claim 1, wherein the TLR is TLR6.
 7. The method of claim1, wherein the cell is derived from one of spleen, thymus, GI tract,lung, kidney, liver, cardiovascular system, blood vessel endothelium,central and peripheral neural system, hematopoietic system, bone marrow,immune system, hair follicle, and reproductive system.
 8. The method ofclaim 7, wherein the cell is derived from the kidney.
 9. The method ofclaim 7, wherein the cell is derived from the spleen.
 10. The method ofclaim 7, wherein the cell is derived from the thymus.
 11. The method ofclaim 7, wherein the cell is derived from the liver.
 12. The method ofclaim 7, wherein the cell is derived from the lung.
 13. The method ofclaim 7, wherein the cell is derived from the immune system.
 14. Themethod of claim 7, wherein the cell is derived from the hematopoieticsystem.
 15. The method of claim 7, wherein the cell is derived from theGI tract.